Inhibition of transfer-messenger RNA aminoacylation and ...Feb 14, 2003 · EF-Tu have RNA chaperone activities, ensuring that tmRNA adopts an optimal conformation during aminoacylation

Running title: aminoglycosides impair trans-translation

Inhibition of transfer-messenger RNA aminoacylation and trans-translation by

aminoglycoside antibiotics.

Sophie Corvaisier, Valérie Bordeau and Brice Felden*

Laboratoire de Biochimie Pharmaceutique, Faculté de Pharmacie, Université de Rennes I,

UPRES Jeune Equipe 2311, 2 avenue du Pr. Léon Bernard, 35043 Rennes, France.

*Corresponding author

Tel: (33)2 23 23 48 51; Fax: (33)2 23 23 44 56; e-mail: [email protected].

Classification: RNA: Structure Metabolism and Catalysis

Abbreviations footnote: tmRNA: transfer messenger RNA; tRNA: transfer RNA; aaRS:

aminoacyl-tRNA synthetase;

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Copyright 2003 by The American Society for Biochemistry and Molecular Biology, Inc.

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Summary

Transfer-messenger RNA directs the modification of proteins whose biosynthesis has stalled

or has been interrupted. Here, we report that aminoglycosides can interfere with this quality

control system in bacteria, termed trans-translation. Neomycin B is the strongest inhibitor of

tmRNA aminoacylation with alanine (Ki value of ∼35 µM), an essential step during trans-

translation. The binding sites of neomycin B do not overlap with the identity determinants for

alanylation, but the aminoglycoside perturbs the conformation of the acceptor stem that

contains the aminoacylation signals. Aminoglycosides reduce the conformational freedom of

the tRNA-like domain of tmRNA. Additional contacts between aminoglycosides and tmRNA

are within the tag reading frame, probably also disturbing reprogramming of the stalled

ribosomes prior protein tagging. Aminoglycosides impair tmRNA aminoacylation in presence

of all the tRNAs from Escherichia coli, small protein B and elongation Factor Tu, but when

both proteins are present, the inhibition constant is one order of magnitude higher. SmpB and

EF-Tu have RNA chaperone activities, ensuring that tmRNA adopts an optimal conformation

during aminoacylation.

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Introduction

In bacteria, transfer-messenger RNA, tmRNA, known alternatively as SsrA RNA or 10Sa

RNA, rescues stalled ribosomes and contribute to the degradation of incompletely

synthesized peptides (for reviews, see 1-3). In a process termed trans-translation, tmRNA

acts first as a tRNA, being aminoacylated at its 3’ end with alanine by alanyl-tRNA

synthetase (AlaRS 4,5) and adding an alanine to the stalled polypeptide chain. Resumption of

translation ensues not on the mRNA on which the ribosomes were stalled but at an internal

position in tmRNA. Translation termination occurs and permits ribosome recycling. Trans-

translation plays at least two physiological roles in bacteria: removing ribosomes stalled upon

mRNAs, and tagging the resulting truncated proteins for degradation.

Because tmRNA is unique to prokaryotes, and is required for viability of some bacteria, it

has attracted the attention of those interested in novel targets for antibiotic therapy. tmRNA

has to be aminoacylated before directing the addition of a peptide tag to the problematic

protein. Moreover, in Neisseria gonorrhoea, and probably also in other bacteria, tmRNA

aminoacylation is essential for resolving stalled translation complexes and preventing

depletion of free ribosomes (6), whereas tagging for proteolysis is dispensable. tmRNA

aminoacylation is therefore an attractive target for blocking trans-translation in pathogenic

bacteria responsible of infectious diseases.

The interactions of aminoglycosides with RNAs represent a paradigm in the use of small

molecules as effectors of RNA function. Aminoglycosides bind and modulate the function of

a variety of therapeutically useful RNA targets (for a review, see 7), and show antimicrobial

as well as antiviral activities. Aminoglycoside antibiotics disrupt the bacterial membrane and

induce miscoding during prokaryotic protein synthesis by binding to the ribosomal A site (8).

They also interfere with translational control (9), with HIV replication (10), inhibit the

activity of several catalytic RNAs by displacing essential metal ions, such as self-splicing

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group I introns (11, 12), hammerhead (13), human hepatitis delta virus (HDV, 14) and hairpin

ribozymes (15) as well as the tRNA processing activity of RNase P RNA (16).

Recent structural (17) and functional (18) evidences indicate that aminoglycosides can

bind and inhibit the aminoacylation of two canonical tRNAs, Escherichia coli (E. coli)

tRNAPhe and yeast tRNAAsp. The crystal structure of yeast tRNAPhe in complex with neomycin

B reveals that the aminoglycoside binds in the deep groove below the D-loop. Inhibition of

aminoacylation of tRNAs is proposed to be either because the aminoglycoside interferes with

the interaction between the aminoacyl-tRNA synthetase (aaRS) and its cognate tRNA through

its binding to major recognition elements (the phenylalanine system; 17) or via a

conformational change of the RNA (the acid aspartic system; 18). For the alanine system,

aminoacylation is determined by a single G3⋅U70 pair (19) and also by minor identity elements

including the discriminator base A73 and a G2⋅C71 pair. This limited set of nucleotides (circled

nucleotides in Fig. 1A-B) is conserved in all the 293 tmRNA genes that have been sequenced

(20). tmRNA is a partial structural mimic of canonical tRNAs thanks to the presence of an

acceptor stem, a T-stem loop and a D analog (21). tmRNA is, however, ∼five fold bigger

compared to canonical tRNAs, contains several pseudoknots, an internal open reading frame,

probably lacks most of the key tertiary interactions present in tRNAs and does not contain an

anticodon stem-loop. Here, we report that several aminoglycosides interact with tmRNA with

affinity and specificity, preventing its aminoacylation with alanine. The tRNA-like domain of

tmRNA is altered upon aminoglycoside binding. Chemical footprints in solution have

explored the structural basis of the interaction between tmRNA and several aminoglycosides.

Trans-translation is therefore a novel target for aminoglycosides.

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Experimental Procedures

Enzymes and RNAs

Alkaline phosphatase and T4 polynucleotide kinase are from New England Biolabs

(Berverly, MA, USA). T4 RNA ligase was from Gibco BRL Life Technologies (Cergy-

Pontoise, France). RNases S1, V1, U2, and T1 were from Amersham-Pharmacia-Biotech

(Orsay, France). [γ-32P]-ATP (3000 mCi/mmol) and [α-32P]-pCp (3000 mCi/mmol) were

from Perkin Elmer (Boston, USA). E. coli tmRNA (22) and tmRNA-TLD (Gaudin et al.,

unpublished results) were over-expressed in E. coli cells and purified as described.

Appropriate bands were electroeluted and pure RNAs were recovered by ethanol

precipitation. Total tRNAs from E. coli are from Sigma-Aldrich (Saint-Quentin, France).

Purified E. coli tRNAAlaUGC was a gift from Dr R. Gillet (MRC, Cambridge, UK).

Aminoacylation experiments

Recombinant alanyl-tRNA synthetase was purified on Ni2+-NTA-agarose (QIAGEN), and

purity was confirmed on a 10% SDS-PAGE. Five independent protein purifications were

required. Final protein concentration ranged from 0.5 to 3.5 µM. Assays were performed at

20 or 37°C in a medium containing 50 mM Tris-HCl (pH 7.5), 10 mM KCl, 20 mM β-

mercapto ethanol, 10 mM MgCl2, 2 mM ATP ([MgCl2]/[ATP] = 5), 0.05 mg/ml of BSA and

42-59 µM L-[14C] alanine (170 mCi/mmol) and 50 to 330 nM of purified E. coli AlaRS.

When varying the pH from 5.5 to 9.5, 50 mM [N-Morpholino]Ethane Sulfonic acid was used

for pH 5.5, and 50 mM Tris-HCl was used for pHs from 6.5 to 9.5. Usually, 1µM of tmRNA

or tmRNA-TLD was denatured 3 min. at 75°C followed by 10 min. at room temperature.

Then, 1µM of purified E. coli (His)6-tagged SmpB, and/or (His)6-tagged EF-Tu.GDP, and/or

EF-Tu.GTP were added and incubated 15 minutes at room temperature. All proteins were at

least 98% pure as judged by SDS-PAGE analysis. EF-Tu.GDP was activated to EF-Tu.GTP

immediately before use by incubation at 37°C for 15 minutes in 50 mM Tris-HCl (pH 7.6), 7

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mM MgCl2, 60 mM NH4Cl complemented with 100 µM GTP, 6 mM phosphoenolpyruvate

(PEP) and 10 µg/ml of pyruvate kinase. After the incubation, the mixture was kept on ice

before use. Various concentrations of aminoglycosides were added, followed by the

aminoacylation buffer, the labeled alanine and the AlaRS. Aliquots were spotted onto 3MM

Whatman papers at different times and trichloroacetic acid precipitated. Kinetic parameters

(KM, Vmax) in the presence and absence of aminoglycosides were performed under steady-state

conditions of enzyme (50-330 nM AlaRS) and substrate concentrations, tmRNA (0.5-3.4

µM) or tmRNA-TLD (0.5-6 µM), and determined from Lineweaver-Burk plots. These

experimental conditions were also applied for tmRNA aminoacylation in the presence of all

tRNAs from E. coli.

Ultraviolet absorbance melting curves

The progressive melting of tmRNA and tmRNA-TLD was monitored by following their UV

absorbency at 258 nm as a function of temperature on a UVIKONXL (Bio-tek instruments)

equipped with a temperature regulator and with a six-cell holder. Temperature was increased

gradually at 0.5°C/min from 15°C to 93°C. Measurements were done in 20 mM potassium

phosphate pH 5.8, 0.5 mM EDTA and 5 mM MgCl2 for tmRNA-TLD and in 20 mM

potassium phosphate pH 5.8, 50 mM NaCl and 5 mM MgCl2 for tmRNA, in the absence and

presence of 500 µM neomycin B, 10 mM tobramycin and 2 mM paromomycin.

Chemical footprints

Labeling at the 5’-ends of tmRNA and tmRNA-TLD were performed with [γ-32P]ATP and

phage T4 polynucleotide kinase on RNA dephosphorylated previously with alkaline

phosphatase. Labeling at their 3’-ends was carried out by ligation of [γ-32P]pCp using T4

RNA ligase. After labeling, tmRNA was gel purified (5% PAGE), eluted, and ethanol

precipitated. Labeled tmRNA is heated 2 min at 80°C and slowly cooled down for 20

minutes at room temperature. Reaction mixtures (15 µl) contained 200 000cpm of [32P]

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tmRNA or tmRNA-TLD and increasing amounts of aminoglycosides (50 to 500 µM of

neomycin B, 0 to 1000 µM of paromomycin and 0 to 6 mM of tobramycin) in 50 mM Hepes

pH 7.5, 25 mM KCl and 2 mM MgCl2. After an incubation of 10 min at room temperature,

ENU, lead-acetate or iron-EDTA were added: 6.25 µl of a solution of ENU saturated in

100% EtOH supplemented with 1 µg of total tRNA, 0.3 mM of lead acetate supplemented

with 2.5 µg of total tRNA and 1 mg/ml of Fe(NH4)2(SO4)2, 5 mM EDTA, 12.5 mM DTT and

0.25% H2O2 supplemented with 2µg of total tRNA for Fe-EDTA mapping (a quick centrifuge

spin mix all four chemicals deposited at four locations inside the eppendorf tube). Incubation

times were 4 hours at 37°C for ENU, 7 minutes at 37°C for lead acetate and 10 minutes at

0°C for Fe-EDTA footprints. RNAs are ethanol precipitated, the pellets are washed twice

with 80% EtOH, dried and counted. Then, the RNA fragments are submitted to 8 or 12%

PAGE. The results were analysed on a PhosphorImager (Molecular Dynamics, Sunnyvale,

CA, USA). During lead mapping of tmRNA in the presence of either tobramycin or

paromomycin, we noticed that gradually increasing the concentration of the aminoglycosides

increases the ratio of the uncleaved versus the cleaved RNA. Modifying the lead

concentration, the incubation times, or the amount of total RNA added during the reaction

could solve the issue. Since the overall conformation of tmRNA changes in the presence of

both aminoglycosides, the conformation of tmRNA in the presence of the aminoglycosides

might be less sensitive to the action of the structural probe.

Results

Inhibition of tmRNA aminoacylation by aminoglycoside antibiotics. Aminoacylation

experiments with purified E. coli alanyl-tRNA synthetase were performed on two RNAs

purified in vivo: tmRNA from E. coli (363 nucleotides, Fig. 1A) and tmRNA-TLD (61

nucleotides, Fig. 1B), a shorter RNA recapitulating the tRNA-like domain of tmRNA.

tmRNA-TLD is capable of being aminoacylated with alanine in vitro (Gaudin et al.,

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unpublished results). The structures of the six aminoglycosides tested are shown in Fig.

1C. Aminoglycosides are subdivided into three major families (23), and representatives of

each family were tested: paromomycin and neomycin B belong to the neomycin family;

amikacin, tobramycin and kanamycin A are members of the kanamycin family, and

gentamicin represents the gentamicin family. All six aminoglycosides inhibit

aminoacylation of tmRNA with alanine, in a concentration dependent manner (Fig. 2). In

presence of each aminoglycoside, the decrease of aminoacylation is not a consequence of

tmRNA or tmRNA-TLD degradation (not shown). Neomycin B is the most efficient

inhibitor, with apparent inhibition constants [Ki] of ∼35 µM for tmRNA-TLD and ∼70 µM

for tmRNA (Fig. 2 and Table I). Neomycin B inhibits the aminoacylation of tmRNA-TLD

with alanine at a ∼30-fold lower concentration than that required to inhibit tRNAAla

aminoacylation (Table I). Paromomycin, gentamicin and amikacin are also potent

inhibitors, whereas kanamycin A and tobramycin are modest ones, with a substantial

difference in activity between the two RNAs (kanamycin A and tobramycin have no

measurable Ki on tmRNA-TLD alanylation). Compared to tmRNA, aminoacylation of

tmRNA-TLD is half inhibited at approximately two-fold higher concentrations of

paromomycin, gentamicin and amikacin (the opposite effect is observed for neomycin B).

Aminoacylation with alanine of a tRNAAla transcript can also be altered by these

aminoglycosides, but the inhibition constants are 3 to 15 fold higher than that of tmRNA.

Amikacin, kanamycin A and tobramycin have related structures (Fig. 1C), although

different inhibition constants (Table I), especially between amikacin and the two others.

Between amikacin and tobramycin, the only differences are at positions R1 and R2 (Fig.

1C). Therefore, the chemical groups substituted at positions R1 and/or R2 of amikacin are

responsible for its lower Ki. Replacing a hydroxyl group (paromomycin) by an amino

group (neomycin B) decreases the Ki for tmRNA three fold (Table I), indicating that a

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minor modification on the chemical structure of an aminoglycoside can increase

significantly its inhibitory activity on tmRNA aminoacylation. All the subsequent

functional and structural investigations were conducted using three representatives,

neomycin B and paromomycin as potent inhibitors and tobramycin as a modest inhibitor of

tmRNA aminoacylation.

The number of charged amino groups is important for inhibition of RNA function by

aminoglycosides. Neomycin B has 6 protonatable aminogroups, their corresponding pKa

are indicated on Figure 1C. In most cases, inhibition of RNA function by aminoglycosides

depends on the number of charged aminogroups (24). tmRNA-TLD inhibition by

neomycin B (at its Ki of 50µM) was monitored at five different pHs (5.5, 6.5, 7.5, 8.5 and

9.5), centered on pH 7.5 that was used in our assays (not shown). At pH 7.5, neomycin B

inhibits 50% of aminoacylation; when pH increases up to 9.5, neomycin B has no

inhibitory effect on aminoacylation (the aminogroups are mostly deprotonated); at pH 8.5,

70% of aminoacylation is detected; when pH decreases to 6.5 and 5.5, inhibition of

aminoacylation by neomycin B is more efficient, leaving only 35% and 20% residual

aminoacylation, respectively. Therefore, inhibition of tmRNA aminoacylation by

neomycin B depends on the number of charged aminogroups, and suggests that complex

formation between tmRNA and neomycin B is mainly driven by electrostatic interactions.

ATP is required during the first step of all the aminoacylation reactions, to form the activated

aminoacyl-adenylate (in our case an alanyl-adenylate). Therefore, aminoglycosides could

bind specifically to the catalytic site of E. coli AlaRS as structural analogs of ATP. To test

this hypothesis, increasing concentrations of ATP, from 1 to 64 mM, were added in the

aminoacylation reaction of tmRNA-TLD with and without 500 µM paromomycin, and in the

aminoacylation reaction of tmRNA with or without 2 mM of tobramycin ([MgCl2] is kept

constant at 20mM). Increasing the concentration of ATP does not relieve the inhibitory effect

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caused by the two aminoglycosides on the aminoacylation of both RNAs (not shown). Thus,

neither paromomycin nor tobramycin function as structural analogues of ATP in the

aminoacylation reaction of tmRNA-TLD and tmRNA with alanine, respectively.

The Michaelis-Menten parameters of tmRNA-TLD and tmRNA aminoacylation with

alanine, in the absence and presence of neomycin B, paromomycin or tobramycin, have been

determined (Table II). In the absence of aminoglycosides, the KM for tmRNA-TLD and

tmRNA are essentially the same (5 to 6 µM) but the Vmax for tmRNA aminoacylation is one

order of magnitude lower than that of tmRNA-TLD (Table II). Compared with tmRNA, the

smaller size of tmRNA-TLD may allow a faster turnover of the substrate during the

aminoacylation reaction. For both RNAs, increasing gradually the concentration of either

neomycin B (15 to 60 µM for tmRNA-TLD), paromomycin (from 50 to 500 µM for tmRNA

and from 100 to 1000 µM for tmRNA-TLD) or tobramycin (from 1 to 3 mM for tmRNA and

from 3 to 10 mM for tmRNA-TLD) increases the KM up to thirty-fold (Table II), but the Vmax

also increases (not shown). Alanylation is not completely inhibited at saturating antibiotic

concentration: there is significant residual aminoacylation level at antibiotic saturation with

tmRNA-TLD and in the presence of amikacin and kanamycin A with tmRNA (Fig. 2). Thus,

the inhibition of tmRNA aminoacylation by aminoglycosides is neither competitive nor non

competitive; therefore the Ki values could not be measured.

Inhibition of tmRNA aminoacylation in the presence of all tRNAs from E. coli. We tested

whether or not aminoglycosides can inhibit tmRNA aminoacylation in the presence of all the

46 tRNAs from E. coli (Table III). For this experiment, we selected paromomycin, at a

concentration corresponding to its apparent inhibition constant for tmRNA alanylation (Table

I). Native tRNAAla isoacceptors represent ∼6 % of the total tRNA population in E. coli (25).

In all the tRNAs from E. coli, up to 60 % of the tRNAAla isoacceptors are able to charge

alanine (100% of charging is not observed probably because our calculated number of

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tRNAAla present in the mixture is overestimated; alternatively, a fraction of tRNAAla might be

degraded). In the presence of all tRNAs from E. coli, there is no inhibition on the

aminoacylation of the tRNA alanine isoacceptors (Table III). This result is consistent with the

fact that purified tRNAAla UGC transcript has a ∼4 fold higher Ki for paromomycin, compared

with tmRNA (Table I). When tmRNA is mixed with all tRNAs from E. coli, paromomycin

decreases the aminoacylation level, even in the presence of a ten-fold excess of total tRNAs

(Table III). Since paromomycin, at that concentration, has no effect on the alanylation of the

tRNAs, the decrease in tmRNA aminoacylation with alanine is responsible for the overall

decrease in the charging levels of the RNA mixture containing both tRNAs and tmRNA.

Thus, paromomycin can inhibit tmRNA alanylation in the presence of all tRNAs from E. coli.

Magnesium ions cannot reverse the inhibitory effect caused by the aminoglycosides.

Positively charged ammonium groups of aminoglycosides match negatively charged metal-

ion binding pockets in RNA three-dimensional structures, displacing divalent metal ions (26).

Recent structural (17) and functional (18) data indicate that the rule also applies for specific

interactions between canonical tRNAs and aminoglycosides. If true for tmRNA, inhibition of

tmRNA aminoacylation by aminoglycosides might be overcome by increasing the

magnesium concentration. The [Mg2+]/[ATP] ratio was kept constant at 5, as in the previous

aminoacylation assays, since it affects the specificity and efficiency of the aminoacylation

reaction (27). The magnesium ion concentration was gradually increased, and the

aminoacylation of tmRNA-TLD and tmRNA, with and without paromomycin and

tobramycin, respectively, was measured (Fig. 3, top). In the presence of paromomycin,

aminoacylation of tmRNA-TLD slightly increases up to 20 mM [Mg2+] whereas in the

presence of tobramycin, aminoacylation of tmRNA show almost no differences up to 10 mM

[Mg2+]. At higher magnesium concentrations, the aminoacylation plateau decreases for both

RNAs with and without aminoglycosides, probably because elevated magnesium

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concentrations start degrading the RNAs (not shown). Therefore, for both RNA-

aminoglycoside interactions, increasing the magnesium concentration during the

aminoacylation reaction cannot rescue the inhibitory effect caused by the aminoglycosides on

aminoacylation. Using lead acetate as a probe, we performed a footprinting experiment

between 500µM neomycin B and labeled tmRNA-TLD at various magnesium concentrations

(1mM, 2mM, 5mM and 10 mM, not shown). The result is that the footprints do not depend

on magnesium concentration, and therefore strengthen our claim that magnesium does not

rescue aminoglycoside inhibition.

Inhibition of tmRNA aminoacylation by neomycin B in the presence of specific ligands.

Small protein B (SmpB) and elongation factor Tu.GTP (EF-Tu.GTP) both enhance alanine-

accepting activity of tmRNA, and SmpB protects the 3’ end of Ala-tmRNA against enzymatic

degradation (28-30). 1µM of tmRNA with post transcriptional modifications charges alanine to

26% on average, but there is a half increase in charging when either 1µM of purified SmpB or

1µM of purified EF-Tu.GTP are present (Fig. 4, table inset). A two-fold increase is observed

when both proteins are present at 1µM. This indicates that in our aminoacylation assays, both

SmpB and EF-Tu.GTP bind tmRNA and enhance its aminoacylation with alanine. With EF-

Tu.GDP, the charging level of tmRNA is not significantly affected, but when in the presence of

SmpB, aminoacylation plateau also increases two-fold (Fig. 4, inset). We verified by native gel

shift assays that SmpB, EF-Tu.GTP and EF-Tu.GDP bind deacylated tmRNA (not shown). In

presence of equimolar ratio of SmpB, neomycin B can still inhibit aminoacylation, but the Ki

increases 1.5-fold, compared with tmRNA alone. In presence of EF-Tu.GTP, the Ki increases

five-fold; in presence of both proteins, the Ki increases ten-fold, compared to tmRNA alone

(Fig. 4). In the presence of 1µM EF-Tu.GDP, the Ki increases 2.5-fold; in the presence of 1µM

SmpB and 1µM EF-Tu.GDP the Ki also increases ten-fold, compared to tmRNA alone (Fig. 4,

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inset). These data indicate that in presence of SmpB and EF-Tu, tmRNA aminoacylation is

significantly protected against the inhibitory effect of aminoglycosides.

Thermal UV melting profiles tmRNA-TLD in the presence of aminoglycosides.

Ultraviolet (UV) absorbance melting curves were performed to assay the effects of

aminoglycosides on tmRNA and tmRNA-TLD structures. In the presence of 5 mM MgCl2,

the melting profile of tmRNA is multiphase, with a first transition around 40°C

corresponding likely to the unfolding of its tertiary structure; a series of smaller transitions,

from 60°C to 90°C, correspond to the progressive unfolding of the secondary structure (not

shown). Since the unfolding pathway of tmRNA is so intricate, no clear picture emerges,

e.g. stabilization or destabilization, when aminoglycosides are present (not shown). For

tmRNA-TLD, however, UV melting experiments have provided information about the

effects of aminoglycosides on the RNA structure (Fig. 5). In the absence of

aminoglycosides, tmRNA-TLD structure unfolds in two transitions; the first one is centred

on a calculated melting temperature (Tm) of around 35°C; the second transition occurs at

higher temperature, with a Tm close to 80°C (Fig. 5A). That second transition is broad and

non-cooperative, suggesting that several conformations of the RNA coexist in solution and

unfold independently when temperature increases. 10 mM tobramycin does not affect the

lower transition, whereas the Tm corresponding to the second transition is considerably

reduced (a decrease of 18°C), to 60°C (Fig. 5B). Also, the second transition becomes

sharper and cooperative. A similar destabilizing effect on tmRNA-TLD structure is

observed in the presence of 2 mM paromomycin, with no significant effects on the lower

transition, but a 16°C drop of the Tm corresponding to the second transition (Fig. 5C). As

for tobramycin, the second transition, whereas shifted to a lower temperature, is

nevertheless sharper and more cooperative, compared to tmRNA-TLD alone. Therefore,

both aminoglycosides reduce the conformational freedom of tmRNA-TLD and stabilize a

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conformation that is no longer aminoacylatable with alanine. In the presence of 500 µM

neomycin B, the Tm corresponding to the first transition is not affected, but the Tm of the

second increases of about 10 degrees, up to 90°C (not shown). Therefore, upon binding

tmRNA-TLD, neomycin B stabilizes its conformation.

Chemical footprints of tmRNA-TLD with neomycin B. To gain structural information about

the interaction between tmRNA-TLD and neomycin B, lead acetate footprints, in the absence

and presence of increasing concentration of neomycin B, were performed (Fig. 3, bottom).

Nine nucleotides (A8-U12, A15-C18) from the D-analog of tmRNA-TLD and nucleotides G31

and A32 are cleaved by lead, but become protected in the presence of increasing concentrations

of neomycin B (Fig. 3, bottom). Six nucleotides involved in the three base pairs U6-A52, G7-

C51 and G34-C50 are cleaved by lead in the absence of the aminoglycoside, suggesting that

these pairs are unstable in tmRNA-TLD; when neomycin B is present, all the six cleavage sites

disappear or are significantly reduced, suggesting that neomycin B, upon binding tmRNA-

TLD, stabilizes the three pairings (result consistent with the increase of the Tm corresponding to

the second transition). There are no visible changes in the conformation of stem-loop H5, the

five upper pairs in H1, the four upper pairs in the T-stem and the T-loop, with and without the

aminoglycoside.

Chemical footprints of tmRNA with paromomycin and tobramycin. The phosphates (ENU)

or nucleotides (lead) of tmRNA that are protected or that become accessible in the presence

of the aminoglycosides are indexed on the right-hand sides of the four upper panels (A-D) in

Figs 6 and 7. The chemical footprints of both aminoglycosides are summarized on the

secondary structure of E. coli tmRNA. Mapping of ribose accessibility with Fe-EDTA shows

no differences in the absence and presence of increasing concentrations of both

aminoglycosides (Figs 6 and 7, panels E are representatives; additional experiments were

performed with longer migration times and 3’-labeling of the RNA to get resolution of the

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upper part of the gels), suggesting that they do not contact any sugars from tmRNA

backbone.

Mapping the phosphates from tmRNA by ENU in the absence of aminoglycoside shows

that out of 363, 39 phosphates are cleaved by the probe and therefore accessible in the native

conformation of tmRNA (Figs 6 and 7, panels A and B). Paromomycin induces 29

concentration dependent chemical footprints onto tmRNA structure, 15 against ENU and 14

against lead (Fig. 6, panels A-D). With lead, two positions that are not accessible in tmRNA

structure, C44 in helix H2 and G150 in the helical portion of pseudoknot PK2, become

reactive in the presence of paromomycin. Due to the conformational flexibility of tmRNA

(22), several nucleotides located in helical portions within the RNA structure are cleaved by

lead in the absence of the aminoglycoside. These nucleotides are U26-U27 (5’ to an internal

bulge within H5), G31-A32 (between two internal bulges in H5, and G31 is involved in GA

pair), U59 (in one stem of PK1), C353 (at one end of H1), U328-U330 (flexible GU pairs in

H5), and G333 (at one end of H5). When paromomycin binds tmRNA, these accessible sites

become protected (direct contact between the aminoglycoside and tmRNA, or indirect effect

of paromomycin on the conformation of tmRNA). Increasing concentrations of paromomycin

modify the reactivity of nucleotides or phosphates located in the tRNA-like domain, H5-PK4,

PK1-H3-H4 and PK2, but not in H6 and PK3. Within the tRNA-like domain of tmRNA,

seven positions (U9, U17, U328-U330, G333 and C353) become protected by paromomycin,

as tobramycin also does but at much higher concentrations (see Fig. 7).

Tobramycin induces 31 concentration dependent chemical footprints onto tmRNA

structure, 14 against ENU and 17 against lead (Fig. 7, panels A-D). As for paromomycin,

C44, in helix H2, become reactive towards lead cleavage. Three nucleotides, G61 in PK1,

G150 in PK2 and G288 in PK4 are protected against both lead and ENU cleavages (Fig. 7,

black stars). The footprints are in the tRNA-like domain, PK1-H3-H4 and in PK2-PK3-PK4.

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Paromomycin and tobramycin share 20 common footprints located, for the most part, in

the tRNA-like domain, the tag reading frame and PK2. Out of the common footprints

between tmRNA and both aminoglycosides, seven are in the tRNA-like domain, indicating

that tight binding requires full-length tmRNA. This result is in agreement with a higher Ki for

tmRNA-TLD aminoacylation by both aminoglycosides, compared with tmRNA (Fig. 2 and

Table I). However, there are significant differences between both aminoglycosides

(underlined nucleotides in Figs 6 and 7): paromomycin, but not tobramycin, modifies

specifically the reactivity of a cluster of nine nucleotides centred on an internal bulge in H5

and in PK4. Tobramycin, but not paromomycin, modifies specifically the reactivity of 11

nucleotides, in PK1, in and around the tag, in PK3 and PK4. Altogether, our probing data

suggest that when aminoglycosides bind tmRNA, there is a significant conformational

rearrangement of the RNA, as already suggested by our UV melting data collected on

tmRNA-TLD.

Discussion

Functional and structural evidence are provided to demonstrate that aminoglycoside

antibiotics interact in vitro with tmRNA from E. coli, modify its conformation in solution,

resulting in its inability to be efficiently aminoacylated with alanine by alanyl-tRNA

synthetase. tmRNA aminoacylation was most strongly inhibited by neomycin B and by

paromomycin followed by, in descending order, gentamicin, amikacin, kanamycin A and

tobramycin (Fig. 2). When tmRNA is reduced to its tRNA-like domain, the concentration of

paromomycin, gentamicin and amikacin required for half inhibition of aminoacylation

increases approximately two-fold (Table I), indicating that structural domains of tmRNA

outside the tRNA-like core are required for optimal inhibition of aminoacylation. This

functional result is confirmed by our structural analysis of tmRNA in complex with two

different aminoglycosides, because paromomycin and tobramycin both induce chemical

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protections of accessible bases and phosphates located outside the tRNA-like domain (Figs 6

and 7). Therefore, with the exception of neomycin B, efficient binding of aminoglycosides

requires full-length tmRNA. Neomycin B interacts specifically with yeast tRNAPhe (17),

rationalizing that the tRNA portion of tmRNA is sufficient for efficient binding.

Between tmRNA-TLD and tRNAAla UGC, the concentration of paromomycin, gentamicin

and amikacin required for half inhibition of aminoacylation increases also approximately

two-fold (neomycin B is an extreme case, with a thirty fold difference). Specific sequences

and/or structural features of tmRNA-TLD, that are not present in tRNAAla, are responsible.

These differences are located in the D-analog and the C21-G30 stem-loop of tmRNA-TLD;

there are also a few sequence variations in the acceptor stem and the T stem-loop of

canonical tRNAAla, compared to tmRNA-TLD. Neomycin B protects accessible nucleotides in

the D-analog of tmRNA-TLD against lead cleavages that are not present in tRNAAla,

rationalizing the binding specificity of the aminoglycoside on tmRNA-TLD. For an

aminoglycoside to inhibit tmRNA aminoacylation, it could prevent the correct recognition of

the limited set of nucleotides that specify the alanine identity, all located in the acceptor stem,

by the AlaRS; alternatively, they could stabilize a non-functional state of tmRNA. Co

crystallization of yeast tRNAPhe with neomycin B reveals that the aminoglycoside is

positioned in the deep groove below the D-loop, possibly interfering with the interaction

between PheRS and its cognate tRNA through its binding to major tRNAPhe charging

determinants (17). Structural data reported here indicate that neomycin B interacts with the D

analog of the tRNA-like portion of tmRNA, but not in the upper portion of the acceptor stem

where the major recognition determinant for alanylation, a GU pair, is located. Upon binding,

neomycin B strengthens three base pairs at the junction between the acceptor and the T

stems, perturbing the conformation of the acceptor stem that contains the aminoacylation

signals. When tmRNA-TLD aminoacylation is performed at 20°C instead of 37°C, the Ki for

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neomycin B increases about four fold (from 35 to 145 µM). Neomycin B is a better inhibitor

when temperature increases, perhaps allowing the RNA to adopt an optimal conformation for

binding.

The footprints of tobramycin and paromomycin on tmRNA and the fact that

aminoglycoside binding can be described in two slopes (at least in some cases, Fig. 2),

suggest that several antibiotic moieties bind to each tmRNA molecule. Both paromomycin

and tobramycin induce the formation of a favoured conformation of tmRNA-TLD, but 7-fold

lower concentrations of paromomycin are required, compared with tobramycin (Table I).

Without aminoglycosides, the conformation of tmRNA-TLD is poorly defined in solution

(Fig. 5), but aminoglycosides trap tmRNA-TLD into an inactive conformation that is either

stabilized (neomycin B) or destabilized (paromomycin, tobramycin). Aminoglycosides

displace a conformational equilibrium towards non-functional tmRNAs; once the

conformational switch is triggered, increasing the magnesium concentration cannot restore

the aminoacylation capacities of tmRNA (Fig. 3).

tmRNA aminoacylation is required for trans-translation. It is not known whether or not

aminoglycosides bind tmRNA in vivo. trans-translation will be impaired in vivo only if the

intracellular concentration of the aminoglycoside, in our case neomycin B, is sufficient to

interact with some of the ∼500 copies of tmRNA per cell (31), in addition to its other RNA

targets. In bacteria, aminoglycosides impair various cellular processes: they mostly disturb

ribosome decoding and cause misreading of the genetic code (32). Paromomycin binds RNA

constructs containing the ribosomal A site with dissociation constants of ∼1.5 µM (33),

indicating that the major target of aminoglycosides is the ribosome. They also inhibit RNase

P RNA cleavage in vitro. The strongest inhibitor of E. coli RNase P RNA cleavage in the

presence of its associated protein is also neomycin B (16), with a 60 µM Ki, (paromomycin

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has a Ki value of 190 µM on RNAse P RNA). Therefore, the Ki values of inhibition of RNase

P RNA cleavage and tmRNA alanylation by both neomycin B and paromomycin are similar.

tmRNA aminoacylation with alanine can be impaired by an aminoglycoside in the

presence of all tRNAs from E. coli (Table III). In its natural context, tmRNA has specific

ligands. Two proteins interact with tmRNA, small protein B (34) and elongation factor-Tu

(35), stimulate tmRNA aminoacylation in vitro (28). When both proteins are added to

deacylated tmRNA prior neomycin B, they prevent tmRNA charging from being inhibited by

low concentrations of aminoglycoside (Fig. 4). Whereas SmpB has a small protecting effect,

EF-Tu, in both the GDP and the GTP forms, has a significant protecting effect against the

inhibition of tmRNA aminoacylation by neomycin B. Protection is maximal when both

SmpB and EF-Tu are present, suggesting that tmRNA aminoacylation might not be impaired

in vivo. RNase T1 footprints of SmpB on a tmRNA transcript indicate that nucleotides G333

(G31 in tmRNA-TLD), G336 (G34 in tmRNA-TLD) are protected by SmpB against RNase

T1 cleavage (28). In presence of neomycin B, G31 from tmRNA-TLD is protected against

lead cleavage and the G34-C50 pairing is reinforced, indicating that there are some overlaps

between the binding sites of SmpB and neomycin B on tmRNA. Deacylated tmRNA forms a

complex with either EF-Tu.GDP or EF-Tu.GTP, and two UV cross-links with EF-Tu.GDP

are located outside the tRNA part, in H5 (U268) and PK4 (U308; 36). Binding sites between

EF-Tu and neomycin B onto tmRNA might overlap. Alternatively, SmpB and EF-Tu could

modify the conformation of deacylated tmRNA, with high efficiency when both proteins are

present, such as tmRNA becomes an efficient substrate for aminoacylation but an inefficient

ligand for neomycin B. In presence of equimolar ratio of purified ribosomal protein S1 that

also interacts with tmRNA, the inhibition constant of tmRNA aminoacylation with neomycin

B increases three-fold (not shown).

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All the known 298 tmRNA gene sequences (20) possess the recognition determinants for

efficient aminoacylation with alanine, suggesting that in all these species the first amino acid

of the tag is alanine. The secondary and probably also tertiary structures of these tmRNA

sequences might be sufficiently conserved to provide the proper recognition determinants for

aminoglycoside binding. Therefore, aminoglycosides are probably also able to interfere with

trans-translation in bacterial species other than E. coli. Mutating the acceptor stem of tmRNA

to confer histidine acceptance retains its ability of protein tagging in vitro, suggesting that the

first alanine of the tag can be substituted by another amino acid (37). Therefore, after specific

mutations within its nucleotide sequence, tmRNA could potentially be chargeable by amino

acids other than alanine. Paromomycin and tobramycin modify the reactivity of the tag

reading frame towards structural probes, likely disturbing re-registration from the stalled

ribosome to the tag and preventing tagging of the truncated proteins, even if aminoacylation

can proceed. The overall pharmacological effect of aminoglycosides during the treatment of

infectious diseases may result from a combination of actions prior and during protein

synthesis, but also when protein synthesis has stalled or has been interrupted.

Aminoglycosides primarily cause misreading of mRNA that leads to the synthesis of

nonsense or truncated peptides. If tmRNA function is also impaired by aminoglycosides, the

nonsense or truncated peptides will accumulate, speeding up cell death.

Acknowledgments

In the lab, we thank Dr L. Metzinger for cloning E. coli SmpB and Dr M. Hallier for

advices on protein purifications. Drs R. Gillet and F. Murphy (MRC, Cambridge, UK) for

critical reading of the manuscript. This work was funded by a Human Frontier Science

Program Research Grant (RG0291/2000-M 100), by a Research Grant entitled “Recherche

Fondamentale en Microbiologie et maladies infectieuses” and by an “Action Concertée

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Incitative Jeunes Chercheurs 2000” from the French Ministry of Research, to B.F. S.C. is

supported by a grant (Projet de Recherche d’Intérêt Régional) from the region of Brittany.

Supplemental data on the chemical footprints

Chemical footprints in solution provided direct evidence about the interaction between

tmRNA and aminoglycosides. The base accessibility of tmRNA was monitored by lead

acetate. Lead acetate cleaves single-stranded RNA, but with sensitivity to subtle

conformational changes of the RNA chain. The accessibility of the riboses was assayed by

Fe-EDTA and Ethyl-Nitroso Urea (ENU) mapped the phosphates. The reactivity towards

these probes was monitored for each nucleotide of tmRNA, with and without increasing

concentrations of either paromomycin (100 to 1000 µM, Fig. 6) or tobramycin (1 to 6 mM,

Fig. 7). Altogether, 24 independent experiments were performed (Figs 6 and 7 are

representative). Mapping the phosphates from tmRNA by ENU in the absence of

aminoglycoside shows that out of 363, 39 phosphates are cleaved by the probe and therefore

accessible in the native conformation of tmRNA (Figs 6 and 7, panels A and B). These

accessible phosphates are scattered throughout the molecule (P7, P13-14, P19, P29, P43, P61,

P64, P66, P87, P90, P97, P100, P108, P135, P151, P157, P195, P197, P200, P204-205, P223,

P226, P228, P274, P288, P293, P297, P305, P308 and P326-333), especially in the upper

portion of H5, around the resume codon and within the four pseudoknots. In the absence of

aminoglycosides, lead acetate cleaves ∼100 nucleotides of E. coli tmRNA (22). With ENU,

several phosphates of tmRNA are not accessible without paromomycin, but become reactive

in the presence of the aminoglycoside. These positions have been omitted, since all are

already reactive in the control lanes without ENU. Nucleotides A69, A79, A86, C91, A94,

G99, A106, A121, A124, U172, A175, C183, A185, A190, A192, A232, U236, A239-U240,

C272, C279, U300 are subjected to cleavage in the presence of the higher concentration (6

mM) of tobramycin; most of them, however, are already present at weaker intensities in the

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control lane with tmRNA alone (Fig. 7). This observation has also been noticed in the

presence of paromomycin, at positions A45, A69, A79 and A174. Therefore, these cleavage

sites are not specific to the interaction and suggest that aminoglycosides can accentuate

cleavage of tmRNA at these sites. Other than these peculiar cases and among the 324

phosphates in tmRNA that are protected against ENU cleavages, none of them becomes

accessible in the presence of tobramycin (Fig. 7).

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Fig. legends

Fig. 1. E. coli tmRNA, tmRNA-TLD (the tRNA-like portion of tmRNA) and the structures

of the aminoglycosides tested in this study (A) Sequence and secondary structure of E. coli

tmRNA (38), with emphasis to the tRNA-like domain; the additional domains containing

four pseudoknots (PK1-PK4) and the tag reading frame are presented in outline (B) E. coli

tmRNA-TLD; both RNAs have an alanine attached to their 3’-ends. Nucleotides

specifying the alanine identity to both RNAs are circled; in grey is shown the GU pair, a

major identity element for alanylation. (C) Structures of the six aminoglycoside antibiotics

used in this study.

Fig. 2. Inhibition of tmRNA (gray circles) and tmRNA-TLD (black squares)

aminoacylation with alanine by six aminoglycoside antibiotics. Aminoacylation plateaus as

a function of increasing concentrations of neomycin B, paromomycin, gentamicin,

amikacin, kanamycin A and tobramycin. For a direct comparison between the two RNAs,

the percentage of charging by AlaRS of tmRNA and tmRNA-TLD without

aminoglycosides was set to 100%, whereas tmRNA is aminoacylated up to 40% and

tmRNA-TLD up to 90%. At low concentrations (100-200 µM) of kanamycin A, a

beneficial effect on tmRNA aminoacylation was reproducibly observed.

Fig. 3. (A-B) Influence of the concentration of magnesium on the aminoacylation of

tmRNA and tmRNA-TLD, in the presence (grey squares) and absence (black diamonds) of

aminoglycosides The MgCl2:ATP is at constant ratio (5:1), while the magnesium

concentration increases. (A) Aminoacylation of tmRNA-TLD in the presence of 500 µM

paromomycin (10% of variance without and with the aminoglycoside). (B)

Aminoacylation of tmRNA in the presence of 2 mM tobramycin (10% of variance without

and with the aminoglycoside). (C-D) Lead acetate footprints of tmRNA-TLD with

increasing concentrations of neomycin B. (C) Autoradiograms of 12% denaturing PAGE of

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cleavage products of 3’-labeled RNAs. Lane C, incubation control; lanes GL, RNase T1

hydrolysis ladder; lanes AL, RNase U2 hydrolysis ladder. The RNA sequence is indexed on

the left side. Gray nucleotides indexed on the right side are the identified protections. (B)

Nucleotides from tmRNA-TLD with a decreased reactivity towards lead cleavage in the

presence of neomycin B are in gray. The major recognition determinant for alanylation, a

GU pair, is boxed.

Fig. 4. Inhibition of tmRNA aminoacylation with alanine by neomycin B in the presence of

purified SmpB, EF-Tu.GTP or both. Aminoacylation plateaus as a function of increasing

concentrations of neomycin B of 1µM tmRNA (T, diamonds), 1µM tmRNA and 1µM

SmpB (T+S, squares) 1µM tmRNA and 1µM EF-Tu.GTP (T+E, triangles) and 1µM

tmRNA, 1µM SmpB and 1µM EF-Tu.GTP (T+E+S, circles). For a direct comparison, the

percentage of charging by AlaRS of tmRNA with and without protein ligands in the

absence of aminoglycoside was set to 100%. Inset table: charging levels and inhibition

constants of aminoacylation of tmRNA with and without SmpB and/or EF-Tu in both the

GDP and the GTP forms. The plots and values reported are averaged from four

independent experiments for each condition.

Fig. 5. Thermal UV melting profiles tmRNA-TLD in the presence of various

aminoglycosides. (A) Thermal UV melting profiles of tmRNA-TLD. The variance in the

calculated Tm was calculated from 13 independent experiments; (B) UV absorbance melting

profile of tmRNA-TLD in the presence of 10 mM tobramycin. The variance in the calculated

Tm was calculated from 6 independent experiments. (C) UV absorbance melting profile of

tmRNA-TLD in the presence of 2 mM paromomycin. The variance in the calculated Tm was

calculated from 6 independent experiments. The first derivative of the UV absorbency as a

function of temperature is also shown. The plots shown are averaged from three independent

experiments.

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Fig. 6. Chemical footprints of increasing concentrations of paromomycin with tmRNA

using ENU (A, B), lead acetate (C, D) and Fe-EDTA (E). Autoradiograms of 8%

denaturing PAGE of cleavage products of 5’ and 3’-labeled tmRNAs, with similar

migration times. Lanes C, incubation controls; lanes GL, RNase T1 hydrolysis ladder; lanes

AL, RNase U2 hydrolysis ladder. Sequence is indexed on the left sides : nucleotides with

black and white triangles are Gs and As, respectively. Nucleotides indexed on the right

sides of the autoradiograms are the identified footprints. Footprints of the aminoglycoside

are indicated onto tmRNA secondary structure, shown schematically. Only the protections

or enhancements of reactivity of nucleotides that vary according to the concentration of the

aminoglycoside were considered as reliable data. Black dots are the phosphates protected

against ENU cleavages in the presence of the aminoglycoside. Grey dots are the

nucleotides protected against lead cleavage in the presence of the aminoglycoside. Black

stars are the positions that are protected by both ENU and lead in the presence of the

aminoglycoside. Grey squares are the nucleotides that become accessible to lead cleavage

in the presence of the aminoglycoside. With ENU, some nucleotides become accessible in

the presence of the aminoglycoside, but all these cleavage sites are already present in the

control lane; thus, they were omitted on purpose. The footprints that are specific to the

aminoglycoside are underlined (those that are common to paromomycin and tobramycin

are not).

Fig. 7. Chemical footprints of increasing concentrations of tobramycin with tmRNA. The

indications provided are identical to Fig. 6.

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Table I. Ki values∗ of the inhibition of aminoacylation of tmRNA, tmRNA-TLD and

tRNAAlaUGC by six aminoglycosides.

RNAs tmRNA tmRNA-TLD tRNAAla UGC

Aminoglycosides

Neomycin B 70 ±20 35 ±15 1000 ±200

Paromomycin 225 ±75 500 ±100 1000 ±100

Gentamicin 400 ±100 700 ±100 1500 ±100

Amikacin 450 ±200 900 ±100 1500 ±100

Kanamycin A 1400 ±500 n.m. n.m.

Tobramycin 1600 ±500 n.m. 2000 ±100

∗Ki (µM) is defined as the concentration resulting in half inhibition of aminoacylation.

Each value reported is the average of 3 to 6 independent experiments, and were

calculated from plots as shown in Fig. 2. n.m.: non measurable. Purified tRNAAla

transcript is the isoacceptor with a UGC anticodon.

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Table II. Kinetic parameters of tmRNA-TLD and tmRNA aminoacylation with alanine in the

presence of aminoglycosides.

RNAs

KM (µM)

Vmax (nM s-1)

tmRNA-TLD

alone 5.5 ±3 3 ±2

+ neomycin B 180 ±20 6 ±3

+ paromomycin 23 ±5 4 ±3

+ tobramycin 26 ±5 3 ±2

tmRNA

alone 6 ±3 0.3 ±.15

+ paromomycin 13 ±3 0.3 ±.1

+ tobramycin 30 ±5 0.4 ±.1

Kinetic parameters represent an average of several experiments using independent enzyme

purifications. The concentrations of the aminoglycosides correspond to their Ki for tmRNA-

TLD and tmRNA aminoacylation with alanine, respectively.

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Table III. Inhibition of tmRNA aminoacylation with alanine by an aminoglycoside in

the presence of all E. coli tRNAs.

Paromomycin - + (250 µM)

picomoles

tmRNA

100

Aminoacylation

37 ± 1

(%)∗

21 ± .9

E. coli tRNAs§

100 (6)

56 ± 8

51 ± 2.7

1000 (60) 52 ± 2 50.5 ± 1

tmRNA with E coli tRNAs§

100 + 100 (6)

100 + 1000 (60)

38 ± .5

41 ± 2

23 ± 1

35 ± 2

∗Charging levels were obtained for 30 min incubation in the presence of equivalent

amounts of purified AlaRS; when both tmRNA and tRNAs were present, charging is

calculated as the percentage of all RNAs aminoacylatable with alanine, without

considering mischarging.

§E. coli tRNAs are all the 46 isoacceptors; the number in parenthesis corresponds to the

amount of isoacceptors tRNAAla in the mixture (25) that can be aminoacylated with

alanine.

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Fig. 1, Corvaisier et al.

A

--•----

G CG CG UG CC GU AG C

ACCA

CGCCCGCGGG

U CA

CT ΨCG

A

A

U

U

C

U

U

G

GA

UCG

A ----

-----

510

15 20

331

345355

363

5’

C GG CGG

3’

24

Tag reading frameResume STOP

PK3PK4

T-loopD-analog

A


ACCA

CGCCCGCGGG

C GG CG C

U CA

A

CU UCG

A

G AA

A

U

U

C

U

U

G

GA

UCG

A

--•----

---

-----

5

10

1520

30

40

45

55

60

5 ’-

3 ’-

_

Acceptorstem

T-loop

T stem

A B

Acceptor stem

T stemD-analog

AlaAla

tRNA-likedomain

PK1 PK2

CR1 R2 R3

O

O

H2N

H O

HN

ON H2

O H

O H

R2

OH O

N H2

R1R3

HH

HH

NH2

OHTobramycinKanamycin A

OH COCHOH(CH2)2NH2 NH2Amikacin

O

O

O

OO

O

OH

OH

OHHOHO

NH2

H2N

NH2

NH2CH2H2N

H

O

O

O

H2N

HON H2

C H3

NHCH3OH

CHR HN R

3

O

NH2

R

NH2

(pKa = 5.7)

(pKa = 7.6)(pKa = 8.8)

(pKa= 7.6)

Gentamicin(C1,C2,C1a)

R = H, CH

HO

HOHO

(pKa = 8.1)HO

RParomomycin

(pKa= 8.6)Neomycin B

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0

20

40

60

80

100

02000

20

40

60

80

100

0

Ala

nyla

tion

of

tmR

NA

and

0

20

40

60

80

100

0 1000 2000 3000 4000

[Kanamycin A] µM

020406080

100

0 200 400 600 800[Gentamicin] µM

tmR

NA

-TL

D (

%)

20

40

60

80

100

00 200 400 600 800 1000

[Amikacin] µM

5000 6000

20

40

60

80

100

00 1000 2000 3000 4000 5000 6000

[Tobramycin] µM

1000

200 400 600 800 1000

[Paromomycin] µM[Neomycin B] µM50 100 150

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[MgCl2] (mM)Cha

rgin

g ef

ficie

ncy

(%)

05 7 8.5

B

20

40

60

80

100

0 10 200

A

Neomycin B (µM)

GLAL C 0 50 100

500

A52C51C50

G34A32G31

C18A15

3’ Lead

CU12U6

15

A


ACCA

CGCCCGCGGG

C GG CG C

U CA

A

CU UCGA

G AA

A

U

U

C

U

U

GA

UCG

A

--•--

---

-----

25

31

5’

_

D-analog

H1

T-loop

H5

32

18G

3’

505152-

-

128 7

6

34


100

80

60

40

20

10

D

G19

A27

G31

A52G53

A58

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0

20

40

60

80

100

0 400 800 1200 1600 2000[Neomycin] (µM)

Cha

rgin

g ef

ficie

ncy

(%)

T

T+S

T+EGTP+S

26 +/-5

38 +/-6

52 +/-6

70 +/-20

115 +/-10

780 +/-100

T+EGTP 35 +/-5 370 +/-90T+EGDP 30 +/-2 180 +/-10

T+EGDP+ S 48 +/-4 700 +/-100

Ki(µM)

Charging levels (%)


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Fig. 5, Corvaisier et al.A

1

34±6

78.5±2.30,004

0,9

0,0030,8

0,7 0,002

Nor

mal

ized

0,650,7

0,750,8

0,850,9

0,951

30 70605040

62.1±1

32±5

d O

D/d

TC

0,4

0,5

0,6

0,7

0,8

0,9

30

0

0,002

0,004

40 50 60 70 80

36±5

Abs

orba

nce

0,60,001

0,5

00,420 30 40 50 60 70 80 90

B1 0,00660.6±1

0,003

0,002

0,001

0

T (°C)

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GLALC

ParomomycinµM

0 100

A45

G90

G126

A86

P61

P90P87

A305G307

G50A45

A323

A327

G321

P97P274P271

P261

P197-P195

P135

500

1000

5’

G50

P100P108

P157

P195-P197

GLALC

ParomomycinµM

0 100

500

1000

G248

3’

P302

P288

GLALC

ParomomycinµM

0 100

500

1000

GL ALC

ParomomycinµM

0 100

500

1000

G13

D

U9

U17

U26-U27G29G31-A32

5’ 3’C353

G333

C44

U59U60

G150

U329U328U330

A B C

A149

A255

A106G310

G293

G29A20

A319

LeadENU LeadENU

Fe-EDTA

GL AL C

ParomomycinµM

0 100

500

1000

5’

G90

G126

A86

5’-3’-

PK1tRNA-- like Domain

PK2

PK3

PK4

H1 H6

H5

H2

H3

P87

P90

P100

P288

U17

U26U27

G29

G31A32

C44

G333

P97

Tagreadingframe

P274

P271 P261

U9C353

P61 U59U60

P108

P302

P197P157

P195

G150

U330U329U328


E

P135H4

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GLALC GLALC 0

TobramycinmM

ENU

0 1 3 6 1 3 6 GLALC 0 1

Lead

3 6

TobramycinmM

5’

G74

G90

G108

G150

A86

A97

A135

P61

P87P90

P100

P157P150

ENU

P97

P108

P197P195

P135

3’

P288

P297

P195

G74

G90

G108

G208

A86

A97

P293

P100-P97P90-P87P61

P150

P197

A255

A305

A273

A207

G293

G248

A323

A273

G310

5 ’ 5’

U60G61

A106

U131

G150

G180

A234

3’

G297G288

C353

G333

U308

G288

A234

A71A70A69

U203

U329U328U330

Lead

TobramycinmM

A B TobramycinmM

C DGLAL C 0 1 3 6

E TobramycinmM

5’

GLALC 0 1 3 6

G90

G126

A86

5’-3’-

PK1tRNA-- like Domain

PK2

PK3

PK4

H1 H6

H5

H2

H3

P87

P90

P100

G288

U17

C44

G333

P97

Tagreadingframe

U9C353

G61U60

P108

P197P157

P195

G150

A69 A70 A71

U308

P135 U131

A106

A234

P297

P293

U203

U330U329U328


G180

H4

Fe-EDTA

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Sophie Corvaisier, Valérie Bordeau and Brice Feldenaminoglycoside antibiotics

Inhibition of transfer-messenger RNA aminoacylation and trans-translation by

published online February 14, 2003J. Biol. Chem.

10.1074/jbc.M212830200Access the most updated version of this article at doi:

Alerts:

When a correction for this article is posted•

When this article is cited•

to choose from all of JBC's e-mail alertsClick here

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Documents

Inhibition of transfer-messenger RNA aminoacylation and ...Feb 14, 2003 · EF-Tu have RNA chaperone activities, ensuring that tmRNA adopts an optimal conformation during aminoacylation